Phosphate crosslinked starch nanoparticle and dental treatments

ABSTRACT

A phosphorous compound such as STMP is used as a cross-linking agent while making a starch nanoparticle in an emulsion process. Negative charge of the nanoparticle is reduced or reversed by adding cations and/or cationizing the starch optionally while forming the nanoparticles. Anionic active agents, such as fluoride or fluorescein, are optionally incorporated into the nanoparticle during the formation process. For example, a fluoride salt can also be used, which promotes the crosslinking reaction while also providing fluoride in the nanoparticle. The retention of both calcium and fluoride in the nanoparticle is improved when both salts are used. Alternatively, the nanoparticle may be used without added calcium and/or fluoride. The nanoparticles may be useful for tooth remineralization, the treatment of dentinal hypersensitivity, to treat caries, or as a diagnostic agent to locate carious lesions.

RELATED APPLICATIONS

This application is a National Stage Entry of International ApplicationNo. PCT/US2019/024619, filed Mar. 28, 2019, which claims priority fromand/or the benefit of U.S. provisional application 62/648,986 filed onMar. 28, 2018 and U.S. provisional application 62/661,669 filed on Apr.24, 2018, both of which are incorporated by reference.

FIELD

This specification relates to biopolymer nanoparticles, for examplenanoparticles containing phosphorous and optionally one or more ofcalcium and fluorine (i.e. fluoride), and to methods of making thenanoparticle. The specification also relates to dental diagnostic andtherapeutic treatments, for example the identification of cariouslesions, tooth remineralization, treatment of carious lesions, ortreatment of dentinal hypersensitivity.

BACKGROUND

International Publication Number WO 2017/070578, Detection and Treatmentof Caries and Microcavities with Nanoparticles, describes nanoparticlesfor detecting and/or treating active carious lesions or microcavities inteeth. The nanoparticle comprises starch bearing at least one cationicregion and/or having a net positive charge and thereby capable ofassociating with carious lesions on a tooth. In some examples thenanoparticles comprise an anticaries agent or a remineralizing agent. Insome examples, the nanoparticles are formed from starch by a reactiveextrusion process as described in U.S. Pub. No. 2011/0042821. After thenanoparticles are formed, they are cationized and fortified with afluoride-containing component, a calcium-containing component or acalcium and phosphate-containing component by lyophilization. Thenanoparticles have a positive zeta potential at the pH of saliva. In oneexample, an anionic fluoride salt was loaded into cationic starchnanoparticles. A 30-minute delay in the release of fluoride inartificial saliva through a dialysis membrane was observed for thenanoparticles relative to a reference solution of free fluoride salt.

INTRODUCTION

The following section is intended to introduce the reader to theinvention and the detailed description to follow but not to limit ordefine any claimed invention.

Dental caries (tooth decay) is the most prevalent chronic disease in theworld. Nearly everyone will develop caries at some point in their life.At any given time 42% of children and 25% of adults have untreatedcaries, leading to complications ranging from pain, infection, poorquality of life and in rare cases, death. Globally it is estimated thatover $200 billion is spent annually on the management of this diseaseand its complications. Surgical treatment results in an irreversiblerestorative cycle leading to several replacement restorations, crownsand eventually tooth loss or dental implants as patients age. Cariouslesions initially form when bacteria in the dental biofilm fermentsugars and produce organic acids, which demineralize enamel. As mineralsleach from enamel rods, the area becomes more porous and weakens. Theearly lesion is comprised of a surface layer (surface zone) whichappears relatively unaffected by the carious attack compared to thesubsurface (lesion body). The surface zone results from mineralprecipitation and is explained by solubility gradients,dissolution/precipitation mechanisms and protection by adsorbed agentspresent in saliva. Reports indicate that the carious lesion body has apore volume (measure of porosity) of 5-30% and the surface zone <5%,compared to sound enamel which has a pore volume of 0.1%. These areas ofsubsurface porosity present clinically as a milky white opacity known asa “white spot lesion”, and identify early stage caries to the clinician.If the process is not reversed, tiny open microchannels in the enamelsurface allow acid to continue entering the subsurface. The lesion thusbecomes more and more porous, until it eventually cavitates, requiringinvasive surgical restoration (dental filling). However, the cariesprocess is dynamic and early stage caries is sometimes reversible withbetter hygiene and remineralization agents such as high fluoridetreatments or toothpastes. Some caries become inactive or “arrested”naturally and do not require any treatment, because the porosityparticularly on the surface has been reduced by mineral and/or proteindeposition. A better understanding of the caries process is leading to aparadigm shift in caries management, which emphasizes enamelpreservation and minimally invasive dentistry, leading to better oralhealth outcomes. However, there is a need for alternative and/orimproved remineralization agents.

Dental caries and other remineralized areas of a tooth are negativelycharged and often found near plaque, which may also be negativelycharged. Phosphorous and calcium are lost when a tooth demineralizes andphosphorous-calcium minerals are useful when remineralizing a tooth.Fluoride is also useful for remineralizing teeth and in the preventionor treatment of caries.

In a process described herein, a phosphorous compound such as STMP isused as a cross-linking agent while making a starch nanoparticle. Thecross-linking agent thereby provides a useful element but STMPcrosslinked nanoparticles have a negative charge and can be expected tobe repelled from caries by electrostatic forces. However, as describedherein, the negative charge can be at least reduced, and optionallyneutralized or reversed, by adding preferably multi-valent cationsand/or cationizing the starch, one or both of which may be doneoptionally while forming the nanoparticles. The addition of a calciumsalt in particular serves to make the charge of the nanoparticle moresuitable for targeting to caries while also providing another element,calcium, that is useful for remineralizing teeth. The addition ofcations such as calcium also appears to increase the retention ofanionic active agents, such as fluoride or fluorescein, which may alsobe incorporated into the nanoparticle during the formation process.Fluoride can usefully react to form minerals in the mouth, or to formminerals with calcium and/or phosphorous in the nanoparticle that can bedelivered to a tooth. Further, a fluoride salt can be used to increasethe ionic strength of the water phase in the nanoparticle formationprocess, which may promote the crosslinking reaction while alsoproviding fluoride in the nanoparticle. While it is not necessary to addeither or both of the calcium salt and the fluoride salt, the retentionof both calcium and fluoride in the nanoparticle is improved when bothsalts are used. Alternatively, the nanoparticle may be used withoutadded calcium and/or fluoride. A nanoparticle with phosphate compoundsas its only remineralizing agents, with the starch cationized such thatthe nanoparticle has a positive zeta potential at pH of 5.5 and under,is also shown to be useful for remineralization. Given that activecarious lesions generally have a pH below 5.5, without intending to belimited by theory, it is expected that the aforementioned nanoparticlewould be beneficial for targeting into the carious lesions, or forpH-triggered targeting if the nanoparticle optionally has a negativezeta potential at a pH of 7 and above. In addition to remineralization,various nanoparticles described herein may be used for the treatment ofdentinal hypersensitivity, to help prevent or treat caries, or as adiagnostic agent to locate carious lesions.

This specification describes methods of making starch basednanoparticles made with a phosphate crosslinker according to an emulsionprocess, and the resulting starch nanoparticles with one or morephosphates. Optionally, the nanoparticles have an anionic active agent,which may be added while making the nanoparticles. Optionally, thenanoparticles have a cation, which may be an active agent, and/orcationic moieties on the starch. The cation and/or cationic moieties maybe added while making the nanoparticles. The nanoparticles may be usedin one or more methods of diagnostic or therapeutic treatment such asthe identification of carious lesions, tooth remineralization, treatmentof carious lesions, or treatment of dentinal hypersensitivity.

In various processes described herein, starch based nanoparticles aremade using an emulsion process such as a phase inversion emulsionprocess. The biopolymer is cross-linked with a phosphate cross-linker,for example STMP. Optionally, one or more of (i) a multivalent cation,such as calcium, (ii) an ionic active agent, such as fluorine (i.e.fluoride) and/or fluorescein, and (iii) one or more starch cationizingagents, are present in the water phase of a water-in-oil emulsion.Compounds may be added to the water phase while the water phase isemulsified (i.e. after phase inversion), during the phase inversion, orbefore the water phase is emulsified (i.e. before phase inversion). Thewater phase also contains the starch and phosphate cross-linker. In someexamples, a fluoride salt and/or a fluorescein salt is added to thewater phase before phase inversion. In some examples, a calcium saltand/or one or more starch cationizing agents are added to the waterphase during or after phase inversion.

Various nanoparticles described herein comprises starch, phosphorous andoptionally one or more active agents such as calcium, fluorine (i.e.fluoride) and fluorescein.

The phosphorous may include one or more starch-phosphate compoundsand/or dangling phosphates. Optionally, the nanoparticles have apositive zeta potential at a pH of 5.5 or less. Optionally, thenanoparticles may have a negative zeta potential at a pH of 7.0 or more.Optionally, the nanoparticles may have a size in the range of 100-700 nmor 100-500 nm as determined by the peak intensity or Z-average size indynamic light scattering (DLS) or as determined by the mean size or D50in nanoparticle tracking analysis (NTA).

This specification also describes a method of providing one or moreelements to a tooth, and the use of a nanoparticle to provide one ormore elements to a tooth. Optionally, the elements are delivered to thetooth in the form of one or more minerals which may be insoluble insaliva at least at ordinary pH, for example 6.2 to 7.6. Optionally, theelements are delivered to a tooth as ions or in salts or other compoundsthat are soluble in saliva at ordinary pH. Optionally, phosphorous isdelivered to a tooth as part of one or more phosphorous-starchcompounds. The nanoparticles may be delivered to the tooth by way ofattachment to plaque on the tooth or by way of attachment to and/orentry into a carious lesion.

This specification also describes the use of nanoparticles to treat atooth, or a method of treating a tooth. The method includes applyingnanoparticles to a tooth. The treatment may provide, for example, one ormore of identification of a carious lesion, remineralization, treatmentof a carious lesion and treatment of dentinal hypersensitivity.

Nanoparticles as described herein can be used to carry phosphorous andoptionally calcium and/or fluorine (i.e. fluoride) to a demineralizedregion of a tooth. The nanoparticles can be applied, for example, byrinsing the mouth with an aqueous dispersion of the nanoparticles.Alternatively, a gel or paste having dispersed nanoparticles therein canbe applied to a tooth. Optionally, some or all of the plaque and/orpellicle can be removed from a tooth before or during the application ofthe nanoparticles, which may help the nanoparticles attach to a carioussurface of the tooth or to enter into pores of the tooth.

However, the removal of pellicle is optional even when targeting cariouslesions. Alternatively, plaque may be left in place and thenanoparticles may be targeted to the plaque.

Without intending to be limited by theory, it is expected that thenanoparticles will help deliver one or more elements to a demineralizedarea of a tooth by one or more of: increasing the concentration of anelement in a rinse, gel or paste relative to a solution ornon-nanoparticle dispersion containing the element; targeting theelements to a demineralized area or associated plaque with or withoutentry of the nanoparticle into a pre-carious lesion; carrying mineralsthat can act as seed crystals, bio-active compounds or filling material;providing one or more elements for reaction with elements released froma tooth or present in saliva; or, providing a delayed or sustainedrelease of one or more elements.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a schematic process flow diagram of biopolymer nanoparticleformation by way of phase inversion emulsion.

FIG. 2 is a chart showing zeta potential values of nanoparticles madeaccording to the process of FIG. 1 at different pH values.

DETAILED DESCRIPTION

Dental cavities begin with one or more areas of demineralization thatpresent as a white spot. The white spot can alternatively be called apre-cavity, a pre-carious lesion or a white spot lesion. The enamel ofthe white spot has greater porosity than natural enamel due to erosiontypically caused by oral bacteria that release acids. The enamel surfacemay remain intact, but in an active lesion open micropores allow acid topenetrate into the tooth. The area of the white spot thus becomes moreand more porous and demineralized over time. Eventually the white spotarea may become so weak that the enamel surface collapses and a cavityis formed. In this specification, references to demineralized areas ofthe tooth include white spots and cavities unless a particular type ofdemineralized area is specified. However, it is expected that thenanoparticles described herein will be particularly useful in treatingwhite spots where it is difficult or damaging to access the porousstructure by conventional means. For example, conventional topicallyapplied fluoride solutions may merely seal the surface of the whitespot, thus making the white spot inactive but not restore the dominantporous area underneath the surface. Drilling the tooth to providefillings or bio-active materials removes some of the natural enamelwhich is then lost forever, as it cannot reform at this stage. Thenanoparticles described herein may be targeted to the white spotsthemselves or to plaque that may be near the white spots. Thenanoparticles described herein may also be useful in occluding orsealing pores in dentin, as in the treatment of dentinalhypersensitivity. It is expected that treatment with the nanoparticleswill be most beneficial when the white spot is in a pre-cavitated stageand active with open micropores and subsurface porosity. However,treatment with the nanoparticles may also be done after a cavity (hole)is formed in more advanced stages of caries.

Without intending to be limited by theory, the inventors believe that ascalcium leaches away from a demineralized area of a tooth, it leavesbehind negative polyphosphate charges in the interior of the tooth.Nanoparticles can enter the demineralized area through micropores in anactive lesion. The concentration of useful elements loaded into ananoparticle dispersion might exceed the concentration of a comparablesolution of the same elements. If the nanoparticles are positivelycharged, they may also associate with the surface (including surfacesbelow the outer enamel surface) of a tooth or plaque as a result ofelectrostatic attraction, thereby increasing either the delivery orretention, or both, of elements in the nanoparticle. Once on or insidethe tooth, saliva degrades the biopolymers and one or more elementsand/or minerals released by the nanoparticle can help restore the poresand/or subsurface area. Materials delivered by the nanoparticles may,for example, react with ions or compounds of the tooth or saliva,provide seed particles for mineral formation, act as a bioactivecompound that can be incorporated into naturally produced minerals, orsimply fill the demineralized subsurface area.

In a method described herein, biopolymer, i.e. starch, nanoparticles aremade using an emulsion process. In brief, one or more biopolymers aredispersed or dissolved in water, the water is then dispersed (i.e.emulsified) in another phase, for example an oil phase, and thebiopolymer is crosslinked while in dispersed droplets of the water phasein the dispersion or emulsion. The use of an oil as a second phase isoptional but helps to load water-soluble reactants into the droplets ofthe water phase. However, another non-solvent of starch, for exampleethanol or hexane, or a multi-phase aqueous system, may be used. Thecrosslinker may be a phosphate or polyphosphate crosslinker such assodium trimetaphosphate (STMP) or sodium tripolyphosphate (STTP).

Optionally, the process may be a phase inversion emulsion (PIE) process.A schematic of a phase inversion process is shown in FIG. 1 . In theexample illustrated, a starch-based nanoparticle is made with a sodiumtrimetaphosphate (STMP) crosslinker. Initially an oil-in-water emulsionis formed which, after an increase in temperature, becomes awater-in-oil emulsion. A surfactant may be used to assist in theoil-in-water to water-in-oil transition and to select the temperature atwhich this transition occurs. The STMP is added so that the crosslinkingreaction occurs within water droplets of the water-in-oil emulsion.Additional elements may be added to the water phase by adding them atany of the three stages shown in FIG. 1 (separate water and oil phases,oil in water emulsion, water in oil emulsion).

Referring to FIG. 1 , an oil phase is homogenized with a water phasecontaining dissolved starch or dispersed starch nanoparticles. The oilmay be, for example, paraffin oil or a food grade mineral oil.Alternatively, other food grade oils such as sunflower oil or olive oilmay be used. After forming an oil in water emulsion, the temperature isincreased to more than the phase inversion temperature (PIT) for thereaction conditions. The PIT may vary depending on the ratio of water tooil, the presence and type of any surfactants (for example Tween 85),the presence and type of any catalysts (for example NaCl) and the typeof oil. In some cases, the PIT may be in the range of 25-60° C.Optionally, heating can be provided by the high shear mixer itself, forexample by increasing the mixer speed to heat the mixture. As the waterin oil emulsion is being heated or after the phase inversion iscomplete, the crosslinker is added. The reaction may then continue, forexample for about an hour.

The biopolymer is crosslinked using a phosphate crosslinker such asSTMP, typically under alkaline conditions. While other crosslinkersmight be used, STMP is advantageously available in food gradepreparations. The crosslinker provides a source of phosphorus, anelement useful for restoring a demineralized tooth. Part of thecrosslinker (the inventors believe the part to be about 10-50% or10-30%) reacts to form internal non-reversible (i.e. covalently bonded)crosslinks within the nanoparticles by way of monophosphate linkage.However, in addition to distarch monophosphate, side reactions may formother compounds such as monostarch triphosphate, monostarchmonophosphate. The reaction is somewhat inefficient but danglingphosphate groups in either inorganic or organic compounds produced inthe reaction or side reactions are available to form a strongassociative complex with calcium and/or fluoride either within theparticle or later when deployed in the oral cavity.

In some examples, NaCl salt is used to provide high ionic strength inthe water phase, which favors the STMP reaction to occur in a subsequentstep. However, in other examples described herein, NaF is used in placeof, or in combination with, NaCl and to also provide fluoride in thenanoparticle. The NaF can be added, for example, in the water phaseproduced prior to homogenizing to form the O/W emulsion in FIG. 1 .Alternatively, the NaF can also be added while homogenizing to form theO/W emulsion or after formation of the O/W emulsion or, though with apossible decrease in fluoride release time, after nanoparticleformation. In some examples, a calcium salt such as calcium chloride isadded in the water phase. Optionally, the calcium salt, dry or inaqueous solution, is added into the W/O emulsion of FIG. 1 after theSTMP is added or, possibly with some decrease in calcium and/or fluoriderelease time, after nanoparticle formation. Water soluble components aredriven into the water droplets and at least partially react or otherwiseassociate with the nanoparticles. In another option, a calcium salt suchas calcium chloride can be added in the water phase produced prior tohomogenizing to form the O/W emulsion in FIG. 1 either in place of NaFand NaCl or in addition to NaF and/or NaCl. STMP produces negativecharges in the resulting nanoparticle. The addition of calcium can blocksome of these charges. However, an additional step such as cationizationof the starch is typically required to produce nanoparticles that arepositively charged at neutral pH or even an acidic pH (i.e 5.5 or less)that may be found within or near a carious lesion.

The fluoride and/or calcium are present with the phosphorous andbiopolymer in a dispersion of small water droplets in an emulsion, forexample a water-in-oil emulsion, optionally stabilized by surfactant.Each droplet containing biopolymer produces a crosslinked particle.Optionally, an emulsion of water in another phase may be used.

The emulsion or emulsions are preferably produced using an ultra-highshear mixer, for example a Silverson dissolver agitator. This mixeradvantageously produces minimal air encapsulation and providessufficient shear to produce nanoparticles averaging under 700 nm orunder 500 nm in diameter most of which, considering their hydrogelnature and distributions in the sizes of the nanoparticles and pores,are able to enter the pores of carious lesions (which average 750-800 nmin size). The starch may be cooked, chemically degraded and/orthermo-mechanically processed to help produce a solution or dispersionof starch in the water phase. Alternatively, smaller starchnanoparticles (20-200 nm) such as those produced by EcoSynthetix Inc.under the trademark EcoSphere™ can be used as the starch feed source.The resultant nanoparticles may have a mean or average size, measuredfor example by the peak in a dynamic light scattering (DLS) plot, theZ-average size (or harmonic intensity averaged particle diameter asdescribed in ISO 13321 or ISO 22412) of a DLS measurement, or the meanor D50 value in a nanoparticle tracking analysis (NTA) measurement, ofless than 1000 nm, for example 100-700 nm, 100-500 nm, 200-500 nm or200-400 nm. After breaking the emulsion, the water phase can optionallybe centrifuged, for example at 4000 rpm for 1 minute, to separate thenanoparticles in the supernatant from unassociated precipitates in thepellet. The nanoparticles are optionally washed to remove traces of oilalthough if a suitable, i.e. food-grade, oil is used it is not necessaryto completely remove all traces of oil. The supernatant can be freezedried to obtain dry nanoparticles. The nanoparticles can be stored dryor, for a more limited time, in an aqueous dispersion, gel or paste. Anaqueous dispersion gel or paste can optionally be sterilized orstabilized with a biocide, antimicrobial preservative or biostaticadditive.

The amount of cross-linker used may be 1% to 50 mol % of STMP based onanhydrous glucose repeating units (AGU). Preferred samples were producedwith 3% to 50% STMP, or from 10% to 50% STMP, for example about 30%STMP. Particle size does not appear to be clearly related to the amountof STMP except that, in some examples, very low amounts of STMP (i.e.1%) produced very small nanoparticles (about 100 nm), low amounts ofSTMP (i.e. 1-5%) produced large nanoparticles (average size of about300-500 nm) while larger amounts of STMP (5% to 50%) producedintermediate nanoparticles (about 100-300 nm). Without intending to belimited by theory, it is possible that samples made with very low STMP(i.e. 1%) do not incorporate substantially all of the available starchinto nanoparticles although 3% STMP seems to be sufficient. Oncesufficient crosslinker is available, the smaller size with largeramounts of STMP may be due to higher crosslinking and less swelling (aspredicted by the Stokes-Einstein equation related to volume swell ratio)since the particles are hydrogels and their size is measured in aswollen state. It is also possible that particle size is influenced moreby the amount of shear energy applied or other factors that could affectdroplet size of the water in oil emulsion. In some cases, nanoparticlesmade with added calcium had zeta potentials near neutral, for example ina range from −5 to +5 mV at a pH of 7.0. Optionally, precipitatesproduced in the water phase that are not associated with thenanoparticles can be separated by centrifugation. The nanoparticles tendto remain in the supernatant of the centrifuged sample. Thenanoparticles exhibit swelling behavior and appear to be hydrogels. Forexample, the nanoparticles retain water, but the amount of waterretained by the nanoparticles decreases with increasing ionconcentration.

The nanoparticles become more negatively charged (as measured by zetapotential) with increasing pH and STMP content. In some examples, thezeta potential of nanoparticles with 1-50 mol % AGU of STMP, withoutcalcium salt added and without starch cationization, ranged from 0 to−65 mV across a range of pH and STMP content, or −10 to −22 mV atneutral pH. For example, samples made with 30% STMP, without calciumsalt added and without starch cationization, were measured as having azeta potential of −15 mV at a pH of 3, −45 mV at pH of 8, and furtherdecreasing to −70 mV at pH of 12.

Adding calcium, for examples as CaCl₂, but still without starchcationization (as described in more detail below) reduces the negativezeta potential of the nanoparticles. At near neutral pH and a 5% STMPcontent, the charge of the nanoparticles with calcium added can be inthe range of −5 mV to 0 mV. Nanoparticles made with calcium and 30% STMPhave a zeta potential in the range of −30 mV to −25 mV near neutral pHand without starch cationization. Without intending to be limited bytheory, the added calcium may be capping the phosphate groups providedby the STMP. Optionally, the starch may be cationized to produce afurther decrease in negative zeta potential, or to produce a positivezeta potential over a desired range of pH.

As an alternative to STMP, sodium tripolyphosphate (STTP) may be used asthe crosslinker.

Optionally, the nanoparticles can be cationized, for example by themethod described in International Publication Number WO 2017/070578,Detection and Treatment of Caries and Microcavities with Nanoparticles.Optionally, the starch may be cationized while in the water in oilemulsion. For example, glycidyl trimethyl ammonium chloride (GTAC),optionally with or pre-mixed with water and isopropyl alcohol or2-proponol, may be added to the water phase before or after forming thewater in oil emulsion. Alternatively, the starch may be cationized afterthe nanoparticles are formed. Alternatively, the starch may becationized before the nanoparticles are formed, although in this casethe starch is preferably first cooked or regenerated so that thecationization is not limited to the surface of the starch granules.

Optionally, a fluorophore could be added to the nanoparticles, forexample by the method described in International Publication Number WO2017/070578, Detection and Treatment of Caries and Microcavities withNanoparticles. Alternatively, a fluorophore can be added during theformation of the nanoparticles, for example by adding the fluorophore tothe water phase. Optionally, the nanoparticles could be co-dispersedwith fluorescent cationic nanoparticles, for example nanoparticlesdescribed in International Publication Number WO 2017/070578, Detectionand Treatment of Caries and Microcavities with Nanoparticles.

The incorporation of PO₄ ³⁻, Ca²⁺ and F⁻ ions (as appropriate) intovarious samples containing phosphate only; phosphate and calcium only;and, phosphate, calcium and fluoride, was confirmed by energy-dispersiveX-ray spectroscopy (EDX/EDS) used for elemental analysis of areas on SEMimages.

The ion content of various freeze dried samples (moisture content lessthan 0.1%) was also measured by inductively coupled plasma (ICP)analysis. The phosphorous content of the nanoparticles increases withthe amount of STMP used. The increase in phosphorous content increasesgenerally linearly with STMP mol %. In one example, nanoparticlesproduced with from 1-50 mol % AGU of STMP had phosphorous contentsranging from about 1000 to about 21,000 ppm. In this example,phosphorous content was determined by ICP-MS after dialyzing the samplesfor 6 days and treating the samples with HCl and HNO₃. The phosphorouscontent values relate to total phosphorous, which is believed to bephosphorous bound to the nanoparticles, but the type of phosphatespecies present was not determined. Optionally, the range of STMP may be3-50 mol % AGU or 10-50 mol % AGU, which gives 4,000-21,000 ppmphosphorous in the nanoparticle. Optionally, the range of STMP may be20-40%, 25-35%, or about 30 mol % AGU.

For example, nanoparticles produced with 30 mol % AGU of STMP butwithout being cationized contained about 15,000 ppm of phosphorous. With67 mol % dry CaCl₂) based on starch (anhydro-glucose repeating units)added, the nanoparticles also contained about 1,320,000 ppm calcium.With 70 mol % NaF and 67 mol % CaCl₂) (based on starch) also added, thenanoparticles contained 19,000 ppm fluoride and calcium contentincreased to 2,160,000 ppm. With 70 mol % NaF (based on starch) addedwithout CaCl₂) (based on starch), the nanoparticles contained about 700ppm fluoride.

The results above indicate that fluoride retention increases whencalcium is also added and that calcium retention increases when fluorideis added. Calcium is believed to interact with phosphate through ionicinteractions. Without intending to be limited by theory, it is possiblethat fluoride can co-precipitate with phosphate and calcium to formfluorapatite (Ca₅(PO₄)₃F) or other minerals in the nanoparticle. It isnot clear if any of the calcium or fluoride is reacted with the starchor phosphates attached to the starch or not, or how the calcium orfluoride are combined with the starch. However, the phosphorous, calciumand fluoride contents described herein are measured in nanoparticlesextracted from the supernatant of a centrifuged sample, whereinprecipitates not bound to the nanoparticles were collected in the pelletof the centrifuge and not part of the measured calcium or fluoridecontents. While no separation is perfect (and so some unboundprecipitates might still be in the nanoparticles), it is expected thatthe combination of phosphorous, calcium and fluoride with thenanoparticle is sufficiently durable for at least a material portion ofthem to be delivered by the nanoparticle to a carious lesion. It is alsoexpected that the addition of a different multivalent cation, forexample a different divalent or trivalent cation or alkaline earth metalion, could similarly increase the retention of anionic active agentssuch as fluoride, though without the potential benefit of addingcalcium.

The nanoparticle might enter the carious lesion through pores in thelesion or stay on the outside of the lesion. Optionally, the teeth maybe cleaned to remove plaque and/or the dental pellicle, for example bypatient brushing or cleaning by a dental hygienist, before or whileapplying the nanoparticles to improve access to a carious lesion. As thenanoparticles break down in saliva while near or inside of the lesion,the phosphorous and calcium and/or fluoride may diffuse into the toothand can deposit or optionally react, with or without additional elementsfrom the tooth or saliva, to form minerals in the tooth. It is alsopossible that already formed minerals, for example calcium phosphate,calciumhydroxyapatite or fluorapatite, present within the nanoparticlescan act as seed crystal or bio-active agents that fill parts of thelesion or are incorporated into additional minerals produced in thetooth.

Optionally the nanoparticle has a zeta potential of at least +2.0 mV,for example between +2.0 mV and +10.0 mV at all pH values of about 7.0or less or at all pH values of about 5.5 or less. In some examples, thenanoparticles have a positive zeta potential in acidic solutions but anegative zeta potential under neutral or basic conditions, i.e. pH of7.0 or more. For example, the zeta potential of the nanoparticles may beat least +2.0 mV at all pH values of about 5.5 or less but negative atpH values of 7.0 or more. Since the pH of saliva in the mouth is roughlyneutral (typically about 7.4), but the pH in or near an active cariouslesion is typically acidic (typically about 4-5), nanoparticles thathave a positive zeta potential only in acidic conditions may be evenmore selectively targeted to active carious lesions than nanoparticlesthat have a positive zeta potential even under neutral or mildly basic(i.e. pH of 7.0 or more) conditions. The nanoparticles are administeredto teeth, for example as a dispersion used in a rinse or mouthwash,which optionally may include diagnostic fluorescent nanoparticles, or ina gel or paste applied to the teeth, or in toothpaste.

The nanoparticles may have a size of up to 2500 nm but preferably have asize of 1000 nm or less. The term “nanoparticles” as used herein is notlimited to particles having a size of 100 nm or less as in the IUPACdefinition but also includes larger particles, for example particles upto 2500 nm, or up to 1000 nm, for example in their largest dimension orin the diameter of a sphere of equivalent volume. Optionally, thenanoparticles may have a mean or average size as determined by peakintensity of a DLS plot, the z-average of a DLS measurement or the meanor D50 of an NTA measurement, in the range of about 100 nm to about 700nm, about 100 nm to about 600 nm, or in the range of about 100 nm toabout 500 nm, or in the range of about 200 nm to about 500 nm, or in therange of about 100 nm to about 400 nm. As mentioned above, particles inthese size ranges will be called nanoparticles, which is consistent withcommon usage of that word in North America particularly for particlesless than 1000 nm in size. However in other parts of the world, andaccording to IUPAC definition, particles larger than 100 nm in size mayalternatively be called microparticles.

Biopolymers, for example polysaccharides and proteins, and in principleany other biopolymer, and mixtures thereof, may be the biopolymer usedin these processes. Any starch, for example waxy or dent corn starch,potato starch, tapioca starch, dextrin, dextran, starch ester, starchether, carboxymethyl starch (CMS), and in principle any other starch orstarch derivative, including cationic or anionic starch, and mixturesthereof, may be the biopolymer used in these processes. Anypolysaccharide, cellulosic polymer or cellulose derivative, for examplemicrocrystalline cellulose, carboxymethyl cellulose (CMC), anynanofibrillar cellulose (CNF), nanocrystalline cellulose (CNC), orcellulose ester, cellulose ether, and in principle any otherpolysaccharide, cellulose or cellulose derivative, and mixtures thereof,may be the biopolymer used in these processes. Proteins, for examplezein (corn protein), casein (milk) or soy protein, and in principle anyother protein or modified protein, and mixtures thereof, may be thebiopolymer used in these processes.

Optionally, the nanoparticles may be prepared by a phase inversionemulsion process as described in U.S. Pat. No. 6,755,915, Method for thePreparation of Starch Particles. In this method starch particles areprepared in a two-phase system comprising steps of a) preparation of afirst phase comprising a dispersion of starch in water, b) preparationof a dispersion or emulsion of the first phase in a second liquid phase,c) crosslinking of the starch present in the first phase, d) separatingthe starch particles thus formed. In some examples the second phaseconsists of a hydrophobic liquid and step b) consists in forming anoil-in-water emulsion. In some examples the second phase consists of awater-miscible non-solvent for starch.

The nanoparticles are stable in dry form. If stored wet, in a closedcontainer, a sterile 5% w/w aqueous dispersion of cationic-mineralcontaining starch nanoparticles, or non-sterile aqueous dispersionstabilized with a citric acid/potassium sorbate or other food-gradebiocide, may be prepared. However, the biocide might not be required.There are some indications of stability without biocide, but it is notyet known whether the nanoparticles have bacteriostatic or bactericidalproperties. Nanoparticles containing fluoride appear to havebacteriostatic or bactericidal properties.

The nanoparticles can be combined with one or more supplemental carriers(i.e. water, excipients or extenders etc.) that are toxicologically andfunctionally acceptable to create a composition that can be administeredto the mount of an animal or person. The composition may be, forexample, a mouth rinse, dentrifice, gel, varnish, paint, toothpaste,tooth powder or mouthwash. The carriers can be selected from the usualcomponents of one or more of these compounds. For example, the carriersmay be one or more of water, alcohols, surfactants, emulsifiers, foamingagents, abrasives, humectants, viscosity modifiers, tackifiers,film-formers, plasticizers, diluents, pH modifiers, sweeteners, flavors,coloring agents and preservatives. For example, the nanoparticles can beused in a rinse or mouthwash that is swished in the mouth and thensuctioned and/or rinsed, at home or in a dental clinic. Alternatively,the nanoparticles can be in a toothpaste administered by brushing theenamel surface at home, or as a paste or gel applied by a hygienist.After being introduced into a mouth, the nanoparticles adhere to plaqueor the surface of caries and may travel inside of active carious lesionsby passing through the porous surface of the lesion.

The nanoparticles can be used, for example, without, or at least withoutan effective amount of, any additional oral care active ingredient suchas an added anticaries agent or remineralizing agent. In particular, thenanoparticles can be used without adding effective amounts of any of theoral care active ingredients described in United States PatentApplication Publication Pub. No. US 2017/0112949 A1 (also published asInternational Publication Number WO 2017/070578, Detection and Treatmentof Caries and Microcavities with Nanoparticles). For example, thenanoparticles can be used without adding, during or after formation ofthe nanoparticle, one or more of a) a fluoride-containing activeingredient, b) a calcium-containing component, c) a calcium andphosphate containing compound and d) amine fluoride, caseinphosphopeptide, phosphoprotein and equivalents and combinations thereof.In this case, the nanoparticle is believed to be effective due to thepresence of one or more phosphate-containing, or starch andphosphate-containing, compounds that are introduced by the addition ofcrosslinker or created in the reaction of the crosslinker and starch.Without intending to be limited by theory, although phosphate alone doesnot create mineral compounds, the phosphate in the nanoparticle mayreact with calcium in saliva or other elements, ions or compounds in themouth to create mineral compounds on or in a tooth or to help nucleatecrystal formation within the white spot or active pre-cavity lesion.

Other two-phase emulsions, for example water and alcohol or hexane,might be used. However, the oil phase helps achieve a high loading ofnon-oil soluble active agents in the nanoparticle. The oil may be a foodgrade mineral oil, or other, preferably food grade, oils such assunflower oil or olive oil. A surfactant, for example Tween 85, is alsoused. The transition temperature may vary depending on the water to oilratio, the type of oil, and the type and amount of surfactant.

The following examples are provided to illustrate various embodimentsand to provide further enabling disclosure but are not intended to limitany claimed invention.

Example 1—Procedure Used for the Preparation of Starch Nanoparticleswithout Cationization in the Emulsion

In a 1 L plastic beaker 30 g of native waxy corn starch was dispersedinto 600 g water along with 16.6 g of NaCl to produce a white suspensionof granules. The dispersion was mixed using a Silverson ultra-high sheardissolver agitator at 6500 rpm. 7.5 g of 50% NaOH was added whilemixing. The mixer was turned up to 8000 rpm and the temperature of themixture increased from 22 to 52° C. in ˜20-30 minutes; at 41-42° C. themixture became transparent; light microscopy of a small droplet placedon a glass slide showed a small fraction of starch granule fragmentspersisted (with birefringence under cross polarizers). At 52° C. allgranule fragments were cooked out in the caustic solution (NaOH servesthe dual purpose of cooking out the starch at lower temps, and ascatalyst for the subsequent crosslinking reaction with STMP). pH papershowed a pH˜13-14. Microscopy showed the final starch solution was fullycooked.

The starch solution was cooled to 19° C. and divided into two equalportions of ˜320 g of liquid; a target of 8.75 g of Tween 80 wasintended, or 29 g of a 30% paste in water, but only 18.53 g was added(all that was left) to one of the two solutions.

Next 300 g (=360 mL; density=0.833) of paraffin oil was added tosolution 1; mixing was started at 3:20 pm.

The droplet size was checked with light microscopy.

Time Temp (° C.) RPM Observations 3:20 22 5500 Oil mixing in OK 3:22 255500 Dispersion noticeably whitens more 3:24 29 5500 O/W to W/O phasetransfer has likely occurred 3:29 36 7500 3:44 48 7500 3:47 52 9000 Add1.5 g TSTP 3:48 54 9500 3:55 64 9500 3:56 65 8500 4:06 71 8500 4:14 738500 4:48 75 8500 End of mixing; cool and neutralize

The final product was a beautiful white suspension. The final mixturewas transferred to capped PE bottle, cooled to <45° C. with cold tapwater, then transferred back to the 1 L plastic beaker, mixed using theSilverson mixer, and ˜300 mL of water was added; a pH probe was mountedand the pH was adjusted from 11.6 to 8.52 using 12 pipette squirts(about 1 mL each) of 1 N HCl. After neutralizing the suspension wasbroken (no longer stable).

Example 2—Production of Nanoparticles, Optionally with Fluoride and/orCalcium, without Cationization in the Emulsion

Cooked 1 L of a 4.68 wt. % starch solution at 55° C. for 2 hrs at pH 13containing 0.3-0.6 M NaCl or NaF and leave to cool. Adjust to a pH of 13obtained by adding NaOH pellets to the water. Alternatively, disperse 60g or thermo-mechanically processed starch (or other cold water solublestarch) in 400 mL deionized water containing 10 g NaCl or molarequivalent of NaF. Either method produces a water phase

Disperse 35 g of Tween 85 in 500 g (600 mL) of paraffin oil to producean oil phase.

Add 400 g of starch solution or dispersion (water phase) to the oilphase.

Homogenize the mixture using a Silverson mixer, keeping track of thetemperature.

Once phase inversion occurs, typically when the temperature is about 50°C., add dry STMP powder if using cooked starch or STMP and 0.95 g NaOHin 3 mL water if using thermo-mechanically processed starch.

Allowed reaction to proceed for 1-4 hr.

Add 200 mL of water containing 3 g 37% HCl for neutralization (=1.5M).

Take a small sample, dilute with 3-5× the volume with water, and checkthe pH using a pH meter. The target pH is 8.0+/−0.5. Adjust pH ifnecessary.

Optionally add dry CaCl₂) powder to remaining sample (while mixing) andmix for 10 mins.

Allow the oil/water emulsion to settle.

Let the dispersion settle without agitation (for example stored in arefridgerator over a weeked). Decanted off a supernatant above any freecalcium-phosphate precipitate. Precipitate nanoparticle in thesupernatant with ethanol. Wash and filter the nanoparticles usingBüchner filtration three times (3×) with ethanol to remove surfactant.

Redisperse the nanoparticles in water and purify by centrifugation or ina separatory funnel (repeat three times) to remove excess oil.

Perform dialysis to remove free phosphate and chloride salts (6 days)and/or centrifuge at 4000 RPM for 1 minute to remove large aggregates ofprecipitate (i.e. CaPO₄) unbound to starch. Freeze dry the sample torecover product nanoparticles.

Example 3—Production of Nanoparticles, Optionally with Fluoride and/orCalcium, with Cationization in the Emulsion

Combine water, starch, sodium hydroxide, surfactant (Tween 85), and oil(mineral oil or paraffin oil) to mixing vessel. Start mixing with highshear mixer and mix until emulsion is formed ˜10 minutes.

Add salt (NaCl) and/or sodium fluoride and/or sodium fluorescein, allowto mix until temperature is >60° C.

To cationize starch, add a mixture of glycidyl trimethyl ammoniumchloride (GTAC), DI-water, and 2-propanol and allow to react for about 1hour (30-90 minutes). Monitor temperature and maintain between 60-75° C.by adjusting high speed mixer mixing rate.

Add STMP to mixture and allow to react/cross-link for about 1-2 hours(30-150 minutes). Monitor temperature and adjust mixing speed tomaintain temperature less than 75° C. but greater than 60° C. Measurebatch pH, and ensure still basic (about pH 10).

If calcium is desired, add calcium chloride (dihydrate), optionallypre-dissolved in water, and mix for an additional 10 minutes.

Neutralize batch (to pH 6.5-8) by addition of HCl if necessary (samplesmade with calcium might not require this step).

Allow sample to cool to room temperature and/or refrigerate tofacilitate phase separation of emulsion.

Dilute sample about 50:50 with 2-propanol and mix. Centrifuge at 10,000RPM for 10 minutes to induce multi-phase separation of particles,aqueous/hydrophilic phase, and oil/hydrophobic phase. Isolateprecipitated particle phase for further purification. Additional rinsingof particles with 2-propanol can help to remove residual oil fromsamples.

Optionally (preferable for samples with calcium), re-disperse particlesin DI-water by shaking, then centrifuge at 1000 RPM for 1-3 minutes toremove any large precipitates, taking the supernatant to the next step.

Flash-freeze the sample and lyophilize to obtain a dry powder sample ofisolated mineral-loaded nanoparticles.

In one example according to the process described above, STMPcrosslinked nanoparticles with calcium are made with: 35 g Tween 85; 500g paraffin oil; 400 g water (initially); 3.125 g NaOH powder; 14.04 gstarch; 13.8 g NaCl; 8.58 g GTAC; 7.22 g 70% IPA; 18 g water (for makinga mixture with GTAC and IPA); 3.38 g STMP; and, 3.4 g CaCl₂*2H₂O.

Example 4—Preparation and Measurements of Nanoparticle Samples withVarious Minerals and Active Agents

Various samples of cationized nanoparticles were made according to themethod in Example 3. The relative amounts of starch and one or more ofphosphate, calcium and fluoride added as a reactant are as shown inTable 1. Amounts of these additives are in pph by mol relative to 100mol of starch AGU. The amount of STMP added while making each sample isone third of the amount indicated in Table 1 to account for the threephosphate groups of each STMP molecule. Fluoride is added by way ofsodium fluoride added during the creation of the initial water phase.Calcium is added by way of calcium chloride added to the water in oilemulsion. A negative control (GMB 9) was provided by nanoparticles madewithout adding any of phosphate, calcium and fluoride. A positivecontrol (GMB 10) was made by dissolution of sodium fluoride in deionisedwater to a concentration of 116 ppm, which matches 1 hour release datafor GMB8 as described below.

TABLE 1 Relative composition of nanoparticle samples GMB1- GMB8 (molsrelative to 100 mols of starch AGU) Sample GMB1 GMB2 GMB3 GMB4 GMB5 GMB6GMB7 GMB8 starch 100 100 100 100 100 100 100 100 phosphate 9 9 9 3 90 9090 90 calcium 0 4.5 0 4.5 0 45 0 45 fluoride 0 0 70 70 0 0 70 70

Calcium release from sample GMB8 and fluoride release from samples GMB7and GMB8 was tested with and without amylase by a dialysis method.Nanoparticles are dispersed in water and placed within a dialysismembrane tube, and measurements are taken periodically by collectingdialysate collected from outside of the dialysis tubes.

With amylase, sample GMB8 released 11 ppm calcium after 1 hour and 21ppm calcium after 72 hours. Without amylase, sample GMB8 released 1 ppmcalcium after 1 hour and 11 ppm calcium after 72 hours. These resultsindicate a slow, time-dependent, release of calcium from nanoparticlescontaining calcium and fluoride.

With amylase, sample GMB7 released 1249 ppm fluoride after 1 hour and21205 ppm fluoride after 72 hours. With amylase, sample GMB8 released116 ppm fluoride after 1 hour and 7604 ppm fluoride after 72 hours.Without amylase, sample GMB7 released 1161 ppm fluoride after 1 hour and21205 ppm fluoride after 72 hours. Without amylase, sample GMB8 released78 ppm fluoride after 1 hour and 8287 ppm fluoride after 72 hours. Theseresults indicate that there was no apparent impact of amylase on therelease time and that both samples show a slow release of fluorideextending beyond one hour. A delayed fluoride release is beneficialsince it can allow a larger dosage of fluoride to be provided safely. Ina home treatment, a delayed release could also make better use ofnanoparticles that become attached to plaque and do not enter thecarious lesions. Whereas fluoride released quickly tends to precipitateat the surface of a lesion, fluoride released slowly may be able toenter through the pores of a lesion before precipitating.

Phosphorous, calcium and fluoride release was also tested by dispersingnanoparticles according to some of the samples from Table 1 incontainers of deionized (DI) water and measuring the concentration ofone of either phosphate, calcium or fluoride in the water at differenttimes up to 2 days. The concentration represents the trend in cumulativerelease of each compound. Calcium was measured by the Arsenazo IIIcolorimetric method. Inorganic phosphorous was measured by the malachitegreen colorimetric method. Fluoride was measured by n ion-specificelectrode using TISAB II.

GMB4, GMB6 and GMB8 were tested for calcium release. GMB4 and GMB8 eachproduced about 1 mg/L of calcium in the water after two days. GMB6produced 3.8 mg/L calcium after 6 hours, 7.8 mg/L calcium after 1 dayand 10.2 mg/L calcium after 2 days.

GMB4, GMB5 and GMB7 were measured for fluoride release. GMB4 produced3.6 ppm fluoride after 1 hour, 40 ppm fluoride after 6 hours, 45 ppmfluoride after 1 day and 51 ppm fluoride after 2 days. GMB5 produced 0.1ppm fluoride after 2 days. GMB7 produced 4.2 ppm fluoride after 1 hour,21 ppm fluoride after 6 hours, 29 ppm fluoride after 1 day and 36 ppmfluoride after 2 days.

GMB3 to GMB 8 were tested for inorganic phosphorous release. GMB3produced 0.49 mg/L phosphorous after 6 hours, 0.55 mg/L phosphorousafter 1 day and 0.50 mg/L phosphorous after 2 days. GMB4 produced 0.37mg/L phosphorous after 6 hours, 0.47 mg/L phosphorous after 1 day and0.64 mg/L phosphorous after 2 days. GMB5 produced 0.81 mg/L phosphorousafter 1 hour, 3.78 mg/L phosphorous after 6 hours, 10.0 mg/L phosphorousafter 1 day and 13.6 mg/L phosphorous after 2 days. GMB6 produced 0.17mg/L phosphorous after 1 hour, 3.41 mg/L phosphorous after 6 hours, 7.29mg/L phosphorous after 1 day and 9.42 mg/L phosphorous after 2 days.GMB7 produced 0.24 mg/L phosphorous after 1 hour, 2.14 mg/L phosphorousafter 6 hours, 4.62 mg/L phosphorous after 1 day and 6.29 mg/Lphosphorous after 2 days. GMB8 produced 0.43 mg/L phosphorous after 1hour, 4.59 mg/L phosphorous after 6 hours, 5.39 mg/L phosphorous after 1day and 6.32 mg/L phosphorous after 2 days.

The average size of the various samples from Table 1 was determined bynanoparticle tracking analysis and dynamic light scattering. Thenumerical average (mean) size of the particles from NTA analysis and theZ-average (intensity based) size from DLS measurements are provided inTable 2. The NTA measurements are believed to be more accurate due tothe polydisperse nature of at least some of the samples. Polydispersesamples are difficult to measure by intensity-based DLS measurements andthe Z-average measurements are skewed towards larger particle sizes.

TABLE 2 Size of nanoparticles Sample GMB1 GMB2 GMB3 GMB4 GMB5 GMB6 GMB7GMB8 NTA size (nm) 525 385 409 477 186 245 148 313 DLS size (nm) 316 756315 341

FIG. 2 shows zeta potential measurements for the samples of Table 1 atdifferent pH values. Samples GMB1 to GMB4, with less phosphorous, arecationic at all pH values tested. Samples GMB6 and GMB8 are cationic atsome pH values but anionic at pH 7. It is expected, due to the similarphosphorous content, that samples GMB5 and GMB7 are also anionic at pH7. This change in charge indicates the potential for “smart” (i.e.pH-triggered) targeting of samples GMB5-GMB8 wherein they would only beelectrostatically attracted to a negatively charged lesion if the lesionhas bacteria actively producing acidic conditions in or near the lesion,while also potentially reducing attraction to proteins or non-specificcellular binding before reaching the lesion. Alternatively, if thenanoparticles are used without first cleaning the teeth (as in atreatment used at home rather than in a dental office procedure),samples GMB5-GMB8 are less likely to be attracted to plaque or organicsmaterial in the mouth that is not near a carious lesion which may benegatively charged but at neutral pH. Optionally, the degree ofcationization of samples GMB1 to GMB4 could be reduced since a zetapotential in the range of +2 to +10 mV is sufficient for targeting tocarious lesions or plaque, or to make samples that are cationic only inacidic conditions.

Example 5—Remineralization Using Non-Cationized Nanoparticles

Nanoparticles were prepared generally as described in Example 2 usingthermo-mechanically processed starch as the starting material to avoidthe step of dissolving native starch granules. The nanoparticles had 20mM of Ca²⁺. Based on TEM images, the nanoparticles appear to have sizesin the range of 200-500 nm and the Z-average size measured by DLS isabout 380 nm. One batch of nanoparticles (GM 6) was prepared with NaCl.Another batch of nanoparticles (GM 6 F−) was prepared with NaF in paceof NaCl.

Six enamel slivers were prepared with a demineralized area on eachsliver. Three slivers were treated with each of nanoparticles GM 6 andGM 6 F⁻ and the results for each nanoparticle were averaged. The sliverswere immersed in dispersions of the nanoparticles for 38 days. SurfaceVMHN measurements were taken at multiple times up to day 16 and thenagain on day 38. The results for GM 6 F⁻ showed a generally linearincrease in hardness from about 75 to about 165 over the first 16 daysand a further increase in hardness to over 200 on day 38. The resultsfor GM 6 showed a generally linear increase in hardness from about 75 toabout 100 and a decrease in hardness to about 75 on day 38. The decreasein hardness on day 38 for GM 6 is believed to be the result of bacterialgrowth on the starch whereas the fluoride of GM 6 F⁻ makes thosenanoparticles antibacterial. Confocal microscopy of cross section of thedemineralized area in the sample treated with GM 6 F⁻ showed a decreasein the lesion depth from 38 μm to 3 μm over the course of the trial.

Example 6—Remineralization with Cationized Nanoparticles

Enamel slivers were prepared with a demineralized area on each sliveraccording to a lactic acid and carboxymethylcellulose (CMC) gel protocolas described in Featherstone et al. (Featherstone, J. D. B. andMellberg, J. R. (1981) Relative rates of progress of artificial cariouslesions in bovine, ovine and human enamel. Caries Res. 15, 109-114). Theenamel slivers were partially protected with an impermeable varnish andsubsequently treated with various nanoparticle formulations as describedin Table 1 while exposed to continued lactic acid and CMCdemineralization for 20 days. The nanoparticle treatment consisted ofcontacting the slivers with one of the nanoparticle samples for 4minutes, 4 times daily, over the course of the 20 days. The continuedlactic acid and CMC demineralization protocol included a 4-hour periodof acid cycling in the aforementioned lactic acid and CMCdemineralization solution and immersion in an amylase-containingartificial saliva solution when the slivers were not immersed in thelactic acid and CMC demineralization solution.

A VMHN value of 300 or more is associated with healthy enamel. A VMHN ofless than 100 is associated with soft demineralized enamel. Table 3gives the average starting and ending VMHN for enamel samples treatedwith each nanoparticle formulation and the controls in the trial. Asindicated by the controls, the trial conditions produce a decrease ofVMHN over time. However, all of the samples GMB1 through GMB8 providedimproved results relative to both controls. Further, treatment withsamples GMB1, GMB4, GMB5, GMB7 and GMB8 showed an increase of VMHN after20 days.

TABLE 3 Initial and 20 day VMHN Sample GMB1 GMB2 GMB3 GMB4 GMB5 GMB6GMB7 GMB8 GMB9 GMB10 Initial VMHN 145 139 126 111 101 114 120 109 112116 20 day VHMN 171 132 96 179 178 87 158 143 24 24

It is interesting to note that GMB5 produced good results but does nothave fluoride or calcium. Without intending to be limited by theory, itis possible that the phosphate groups of this sample draw calcium fromsaliva to produce precipitates in the tooth.

Example 7—Treatment of Dentinal Hypersensitivity

Two oral gels were formulated with nanoparticles according to sampleGMB8 described above. The gels were stabilized with propylene glycol andthickened with food grade gelatin. Formulation 1 had 25% w/w of thenanoparticles. Formulation 2 had 5% w/w of the nanoparticles. The effectof the two gel formulations containing on dentinal hypersensitivity wasdetermined by applying the formulations to dentine discs and measuring achange in dentinal permeability as indicated by measurements ofhydraulic conductance. Since it is believed that dentinalhypersensitivity (DH) involves neural stimulation by fluid flow withindentinal tubules, occlusion of the tubules as indicated by a change inhydraulic conductance indicates a potential treatment of DH.

The dentine discs were cut from extracted human third molar teeth. Eachtooth was cut transversely just below the widest part of the tooth, thensectioned to produce dentine discs with a thickness of 0.5 mm. Each dischas a minimum 4 mm diameter. The discs are polished with carbide paperuntil flat and then acid etched to remove any smear layer producedduring the cutting and polishing procedures. The disc is placed in atube and water is applied against the disc at a pressure of 1 psi. Thevolume of water passing through the disc in 5 minutes is measured. Thetest is performed before and after treatment of the disc with thenanoparticles and again after storing the samples for 24 hours inartificial saliva. The treatments are done by: soaking the discs inartificial saliva for 30 minutes; applying one of the gel formulationsto each disc; rubbing the gel on the dentin surface for 2 minutes;waiting for 10 minutes; and, rinsing the discs.

Samples treated with nanoparticles according to formulation 1 showed areduction in flow (relative to flow before treatment) of about 40%immediately after application of the nanoparticles and after 24 hours.Samples treated with nanoparticles according to GMB8 formulation 2showed a reduction in flow of about 75% immediately after application ofthe nanoparticles and after 24 hours.

Example 9—Fluorescent Nanoparticles

Sodium fluorescein and calcium chloride were added to a vial of water ata pH of about 10. A precipitate of calcium fluorescein formed at thebottom of the vial.

Nanoparticles were made according to the process described in Example 3with: 74 g Tween 85; 1121 g mineral oil; 842 g water (initially); 13 gNaOH powder; 39 g starch; 29 g NaCl; 0.75 g sodium fluorescein; 25 gGTAC; 20 g 70% IPA; 50 g water (for making a solution with GTAC andIPA); 28 g STMP; and, 27 g CaCl₂*2H₂O pre-dissolved in 200 g water.

The resulting nanoparticles had a yellow-orange color indicating thepresence of fluorescein in the nanoparticles. The nanoparticles may beused for the purposes described for starch nanoparticles reacted withfluorescein amine coupled onto the starch nanoparticles via EDC-NHSchemistry as described in described in United States Patent ApplicationPublication Pub. No. US 2017/0112949 A1 (also published as InternationalPublication Number WO 2017/070578), Detection and Treatment of Cariesand Microcavities with Nanoparticles. However, the method of makingfluorescent nanoparticles described herein is less expensive and thenanoparticles simultaneously deliver beneficial agents to the caries.

International Publication Number WO 2017/070578, Detection and Treatmentof Caries and Microcavities with Nanoparticles, is incorporated byreference. International Publication Number WO 2013/081720 A1, AptamerBioconjugate Drug Delivery Agent, is incorporated herein by reference.All of the patent publications and other publications mentioned hereinare incorporated by reference.

The term “preferable” or variants thereof indicates that something ispreferred but optional. Words such as “may” or “might” are meant toinclude the possibility that a thing might, or might not, be present.The words “fluorine” and “fluoride” are intended to include whateverform of fluorine may be present. For example, the word “fluoride” is notintended to require the presence of distinct, soluble F⁻ ions, but couldbe satisfied by the presence of fluorine in the insoluble part of acompound such as calcium fluoro apatite, in combination with a salt suchas CaF₂ whether the salt is in a dissolved or precipitated state, orcombined with the nanoparticle in any other way.

We claim:
 1. Nanoparticles comprising starch and phosphorous, whereinthe nanoparticles have a positive zeta potential at a pH of 5.5 or less,wherein the nanoparticles have a mean or average size in the range of100-500 nm as determined by the Z-average size in dynamic lightscattering (DLS) or as determined by the mean size in nanoparticletracking analysis (NTA).
 2. The nanoparticles of claim 1 comprising oneor more of fluoride, fluorescein or calcium.
 3. The nanoparticles ofclaim 1 comprising fluoride and calcium.
 4. The nanoparticles of claim 1having a negative zeta potential at a pH of 7.0 or more.
 5. Thenanoparticles of claim 1 incorporated into an aqueous dispersion, a gelor a paste.
 6. The nanoparticles of claim 1 wherein the phosphorous ispresent in starch-phosphate compounds and/or dangling phosphates.
 7. Amethod of making nanoparticles of claim 1 comprising the steps of,preparing a first phase comprising a solution or dispersion of starch inwater; preparing a dispersion or emulsion of the first phase in a secondliquid phase such as an oil phase; adding one or more of (a) one or moremulti-valent cations, and (b) one or more starch cationizing agents tothe first phase; and, crosslinking the starch in the first phase with aphosphate crosslinker.
 8. The method of claim 7 comprising adding ananionic active agent to the first phase, wherein the anionic activeagent comprises fluoride.
 9. The method of claim 7 comprising adding ananionic active agent to the first phase, wherein the anionic activeagent comprises fluorescein.
 10. The method of claim 7 wherein the oneor more multivalent cations comprises calcium.
 11. The method of claim 7comprising adding one or more starch cationizing agents.
 12. The methodof claim 7 wherein the crosslinker comprises sodium trimetaphosphate.13. A method of filling a tooth or supporting the remineralization of atooth comprising administering nanoparticles of claim 1 fortified withcalcium and/or fluoride to the tooth.
 14. The method of claim 13 whereinthe nanoparticles are dispersed in a liquid, paste or gel carrier andapplied to the tooth.
 15. The method of claim 14 wherein thenanoparticles are applied to a tooth after or while cleaning a pellicleand/or plaque from the tooth.
 16. The method of claim 13 wherein thenanoparticles comprise a mineral, for example calcium phosphate,fluorapatite or calcium hydroxyapatite.